Proteins with cardioprotective activity

ABSTRACT

A protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and a fragment thereof, or a polynucleotide encoding therefor, for use in treating or reducing the risk of heart disease.

FIELD OF THE INVENTION

The present invention relates to the use of Chrdl1, Fam3c and Fam3b asmedicaments, for example in the context of gene therapy or throughadministration as proteins, such as recombinant or synthetic proteins,for treating or reducing the risk of heart disease. In particular, theinvention relates to protection of the heart against the development ofheart failure (HF) by preserving cardiac muscle cell viability.Conditions for which the medicaments are effective include, but are notlimited to, cardiac ischemia (myocardial infarction and reperfusioninjury), cardiac toxic damage and cardiomyopathy of genetic origin.

BACKGROUND OF THE INVENTION

Despite recent advances in cardiovascular surgery and therapy,cardiovascular disorders (CVDs) still account for ˜30% of deathsworldwide, of which approximately 50% are due to ischemic heart disease,with a trend to increase to over 23 million by 2030, according to theWHO (www.who.int/cardiovascular_diseases/en/). Coronary artery diseaseand myocardial infarction represents the major cause (65-70% of cases)of heart failure. The prognosis of this condition remains poor, withmortality estimated at 40% of patients at 4 years from diagnosis (Owan,T. E., et al. Trends in prevalence and outcome of heart failure withpreserved ejection fraction. N Engl J Med 355, 251-259 (2006)). Anessential component underlying the epidemic burden of HF is theinability of the cardiac muscle to undergo regeneration in adult life.Cardiac injury, as a consequence of ischemia, hypertension, infection,inflammation or toxic damage, typically results in irreversible loss ofcardiomyocytes (CMs), with consequent fibrosis and scarring (Laflamme,M. A. & Murry, C. E. Heart regeneration. Nature 473, 326-335 (2011);Xin, M., Olson, E. N. & Bassel-Duby, R. Mending broken hearts: cardiacdevelopment as a basis for adult heart regeneration and repair. Nat RevMol Cell Biol 14, 529-541 (2013)).

Drug development in this area has been marginal in terms of efficacysince the mid 1990s, with all the available drugs being small chemicalmolecules. If one considers the current ESC guidelines for chronic HFwith reduced ejection fraction (Ponikowski, P., et al. 2016 ESCGuidelines for the diagnosis and treatment of acute and chronic heartfailure: The Task Force for the diagnosis and treatment of acute andchronic heart failure of the European Society of Cardiology (ESC). Eur JHeart Fail 18, 891-975 (2016)), the three drugs recommended in allpatients (ACE inhibitors, β-blockers and mineralocorticoid/aldosteronereceptor antagonists) have all been introduced in clinical practice inthe 1970s or before (Gavras, H., Faxon, D. P., Berkoben, J., Brunner, H.R. & Ryan, T. J. Angiotensin converting enzyme inhibition in patientswith congestive heart failure. Circulation 58, 770-776 (1978); Swedberg,K., Hjalmarson, A., Waagstein, F. & Wallentin, I. Prolongation ofsurvival in congestive cardiomyopathy by beta-receptor blockade. Lancet1, 1374-1376 (1979); Goldberger, E. Aldosterone and the Edema ofCongestive Heart Failure. Am J Cardiol 15, 274 (1965)); of the drugsonly recommended in selected patients, angiotensin II receptorblockers-ARBs date back to the mid 1990s (Gottlieb, S. S., et al.Hemodynamic and neurohormonal effects of the angiotensin II antagonistlosartan in patients with congestive heart failure. Circulation 88,1602-1609 (1993)), while the more recent LCZ6969 is based on acombination of an old ARB (valsartan) with sacubitril, belonging to theneprilysin inhibitors class, which were also developed in the late 1980s(Jhund, P. S. & McMurray, J. J. The neprilysin pathway in heart failure:a review and guide on the use of sacubitril/valsartan. Heart 102,1342-1347 (2016)). The last drug in the recommendations, ivabradin, anI_(I)-channel inhibitor that controls heart rhythm, was also developedin the mid 1990s (Thollon, C., et al. Electrophysiological effects of S16257, a novel sino-atrial node modulator, on rabbit and guinea-pigcardiac preparations: comparison with UL-FS 49. Br J Pharmacol 112,37-42 (1994)).

Studies on candidate biological factors for these conditions indicatedby biochemical studies (e.g. relaxin, natriuretic peptides, AVPantagonists) have all failed in phase III clinical trials (Teerlink, J.R., et al. Serelaxin in addition to standard therapy in acute heartfailure: rationale and design of the RELAX-AHF-2 study. Eur J Heart Fail19, 800-809 (2017); O'Connor, C. M., et al. Effect of nesiritide inpatients with acute decompensated heart failure. N Engl J Med 365, 32-43(2011); Matsuzaki, M., Hori, M., Izumi, T. & Fukunami, M. Efficacy andsafety of tolvaptan in heart failure patients with volume overloaddespite the standard treatment with conventional diuretics: a phase III,randomized, double-blind, placebo-controlled study (QUEST study).Cardiovasc Drugs Ther 25 Suppl 1, S33-45 (2011); Wang, G., et al.Efficacy and Safety of 1-Hour Infusion of Recombinant Human AtrialNatriuretic Peptide in Patients With Acute Decompensated Heart Failure:A Phase III, Randomized, Double-Blind, Placebo-Controlled, MulticenterTrial. Medicine (Baltimore) 95, e2947 (2016)).

In particular, no drug or treatment of any kind protects the heartduring acute ischemia, as well as after myocardial infarction. When apatient undergoes myocardial infarction, cardiac cells progressively diebecause of the sudden lack of oxygen due to the blockage of a coronaryartery. If a patient is revascularized (percutaneous coronaryintervention, angioplasty) in the first hours after infarction, asignificant portion of the myocardium is spared, however still a largenumber of myocardial cells irreversibly undergo death. Angioplastyitself promotes additional damage, due to the sudden flux of oxygenoccurring after restoring blood perfusion. Due to the incapacity ofcontractile myocardial cells to undergo significant regeneration in theadult life, the lost portion of the myocardium is irreversibly repairedthrough formation of a scar, which, in the long term, is a majordeterminant of heart failure.

Therefore, there is still the strongly felt need to provide a drug, inparticular a biological drug mimicking endogenous survival processes,that could save myocardial cells immediately after an insult causingloss of cardiomyocytes and consequent pathological remodeling of theheart. In particular, this need is relevant for the treatment of severalconditions leading to HF, including myocardial infarction,reperfusion-injury after angioplasty, cardiac toxic damage by cancerchemotherapy, myocarditis, cardiomyopathy of genetic and non-geneticorigin.

Protecting cardiomyocytes from death would be of immense relevance,since it would permit sparing the myocardium and allowing long-termpreservation of cardiac integrity and function, avoiding the occurrenceof deterioration of cardiac function leading to HF.

Despite the lack of curative therapies, notable progress has beenachieved in understanding the cellular and molecular mechanisms leadingto tissue degeneration.

Thus, there is also the need for novel biological therapeutics, able tospecifically interfere with the different mechanisms of disease onsetand progression, offering therapeutic opportunities.

Since the approval of recombinant insulin in 1982 (Humulin®), the numberof biotechnological drugs has increased exponentially in the last threedecades. If one considers monoclonal antibodies, enzymes, receptormodulators, subunit vaccines and peptides, well over 350biotechnological drugs have now gained clinical approval and over 400have entered clinical trials (Kinch, M. S. An overview of FDA-approvedbiologics medicines. Drug Discov Today 20, 393-398 (2015); Rader, R. A.(Re)defining biopharmaceutical. Nat Biotechnol 26, 743-751 (2008)).

In genetic research, several of the ground-breaking discoveries havebeen obtained through screening approaches. Starting in the 1980s, geneidentification has been initially facilitated by the selection ofgenomic DNA libraries by hybridization, followed by the identificationof cDNAs by antibody screening in phage libraries. In the late 1980s andin the 1990s, functional screening of libraries in cultured cells led tothe identification of several oncogenes and cellular receptors foranimal viruses. Most early approaches were based on the use of pooledlibraries (typically, cDNA libraries), in which a desired factor wasidentified by phenotype-based selection. In the 2000s, along withadvancements in robotics, library screening progressively moved towardhigh throughput screening (HTS) analysis, based on the use of arrayedlibraries. HTS has paved the way to the use not only of cDNA libraries,but also of libraries of peptides, nucleic acids (Eulalio, A., et al.Functional screening identifies miRNAs inducing cardiac regeneration.Nature 492, 376-381 (2012)) and small molecules. Today, the advances ingene transfer permit the field to take a further step forward, namely toscreen libraries directly in animals, thus moving from biochemical orphenotypic selection in vitro towards true functional selection in vivo.

SUMMARY OF THE INVENTION

The inventors have utilized a unique procedure based on the in vivoFunctional Selection (FunSel) of factors exerting a desired function toidentify factors with cardioprotective effects. The inventors' procedureis based on the use of a library of AAV vectors, which are exquisitetools for highly efficient cardiac gene transfer.

Of note, factor identification by FunSel does not require that theselected factors play a role in a given tissue during normal physiology,thus extending the range of potentially therapeutic proteins to allsecreted factors encoded by the genome.

The inventors have surprisingly found that the three factors Chrdl1(Chordin-like protein 1), Fam3c and Fam3b, although exerting completelydifferent biological activities, commonly share cardioprotectiveeffects.

The three novel cardioprotective proteins Chrdl1, Fam3c and Fam3b arehighly homologous between mouse and humans (93%, 94% and 79%respectively).

These three factors protect from cardiac cell death induced by ischemiaand other kind of damage, including treatment with chemotherapeuticagents.

Accordingly, the present invention relates to the factors Chrdl1, Fam3cand Fam3b for use as medicaments.

In one aspect, the invention provides a protein selected from the groupconsisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or apolynucleotide encoding therefor, for use in treating or reducing therisk of heart disease.

In another aspect, the invention provides Chrdl1 or a fragment thereof,or a polynucleotide encoding therefor, for use in treating or reducingthe risk of heart disease. In another aspect, the invention providesFam3c or a fragment thereof, or a polynucleotide encoding therefor, foruse in treating or reducing the risk of heart disease. In anotheraspect, the invention provides a Fam3b or a fragment thereof, or apolynucleotide encoding therefor, for use in treating or reducing therisk of heart disease.

In another aspect, the invention provides a method for treating orreducing the risk of heart disease, wherein the method comprisesadministering a protein selected from the group consisting of Chrdl1,Fam3c, Fam3b and fragments thereof, or a polynucleotide encodingtherefor, to a subject in need thereof.

In some embodiments, the use reduces the risk of heart failure. In someembodiments, the risk of heart failure is reduced in a subject sufferingfrom a heart disease. In some embodiments, the risk of heart failure isreduced in a subject at risk of heart disease.

In one aspect, the invention provides a protein selected from the groupconsisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or apolynucleotide encoding therefor, for use in reducing the risk of heartfailure.

In another aspect, the invention provides Chrdl1 or a fragment thereof,or a polynucleotide encoding therefor, for use in reducing the risk ofheart failure. In another aspect, the invention provides Fam3c or afragment thereof, or a polynucleotide encoding therefor, for use inreducing the risk of heart failure. In another aspect, the inventionprovides a Fam3b or a fragment thereof, or a polynucleotide encodingtherefor, for use in reducing the risk of heart failure.

In another aspect, the invention provides a method for reducing the riskof heart failure, wherein the method comprises administering a proteinselected from the group consisting of Chrdl1, Fam3c, Fam3b and fragmentsthereof, or a polynucleotide encoding therefor, to a subject in needthereof.

In some embodiments, the risk of heart failure is reduced in a subjectsuffering from a heart disease. In some embodiments, the risk of heartfailure is reduced in a subject at risk of heart disease.

In another aspect, the invention provides a protein selected from thegroup consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or apolynucleotide encoding therefor, for use in preserving cardiac musclecell viability.

In another aspect, the invention provides Chrdl1 or a fragment thereof,or a polynucleotide encoding therefor, for use in preserving cardiacmuscle cell viability. In another aspect, the invention provides Fam3cor a fragment thereof, or a polynucleotide encoding therefor, for use inpreserving cardiac muscle cell viability. In another aspect, theinvention provides Fam3b or a fragment thereof, or a polynucleotideencoding therefor, for use in preserving cardiac muscle cell viability.

In another aspect, the invention provides a method for preservingcardiac muscle cell viability, wherein the method comprisesadministering a protein selected from the group consisting of Chrdl1,Fam3c, Fam3b and fragments thereof, or a polynucleotide encodingtherefor, to a subject in need thereof.

In some embodiments, cardiac muscle cell viability is preserved in asubject suffering from a heart disease. In some embodiments, cardiacmuscle cell viability is preserved in a subject at risk of a heartdisease.

An example amino acid sequence of Chrdl1 is SEQ ID NO: 1. An examplenucleotide sequence encoding Chrdl1 is SEQ ID NO: 4.

An example amino acid sequence of Fam3c is SEQ ID NO: 2. An examplenucleotide sequence encoding Fam3c is SEQ ID NO: 5.

An example amino acid sequence of Fam3b is SEQ ID NO: 3. An examplenucleotide sequence encoding Fam3b is SEQ ID NO: 6.

In some embodiments, the protein comprises an amino acid sequence thathas at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identityto SEQ ID NO: 1. In some embodiments, the protein comprises an aminoacid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identity to SEQ ID NO: 2. In some embodiments, the proteincomprises an amino acid sequence that has at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3.

In some embodiments, the protein comprises the amino acid sequence ofSEQ ID NO: 1. In some embodiments, the protein comprises the amino acidsequence of SEQ ID NO: 2. In some embodiments, the protein comprises theamino acid sequence of SEQ ID NO: 3.

In some embodiments, the polynucleotide comprises a nucleotide sequencethat has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 4. In some embodiments, the polynucleotidecomprises a nucleotide sequence that has at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5. In someembodiments, the polynucleotide comprises a nucleotide sequence that hasat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity toSEQ ID NO: 6.

In some embodiments, the polynucleotide comprises the nucleotidesequence of SEQ ID NO: 4. In some embodiments, the polynucleotidecomprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments,the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6.

The heart disease may, for example, be due to loss of cardiomyocytes asa consequence of myocardial infarction, reperfusion-injury afterpercutaneous coronary intervention (coronary angioplasty), hypertension,cardiac toxic damage (in particular, by cancer chemotherapy),myocarditis, cardiomyopathy of genetic and non-genetic origin.

In some embodiments, the heart disease is associated with cardiacischemia. In some embodiments, the heart disease is associated with lossof cardiomyocytes.

In some embodiments, the heart disease is selected from myocardialinfarction; the consequences of myocardial infarction;reperfusion-injury after percutaneous coronary intervention (coronaryangioplasty); myocarditis; hypertension; cardiac toxic damage (inparticular, by cancer chemotherapy); or cardiomyopathy.

In some embodiments, the heart disease is ventricular dysfunction.

In some embodiments, the heart is protected from myocardial infarction.In some embodiments, cardiac function is preserved after myocardialinfarction or percutaneous coronary intervention (coronary angioplasty).In some embodiments, fibrosis after infarction is reduced.

In some embodiments, the protein or polynucleotide results in heartprotection from myocardial infarction and/or other conditions leading toloss of cardiomyocytes, preferably preserving cardiac function andreducing reparative fibrosis.

In some embodiments, heart failure is prevented.

In some embodiments, the protein is administered by direct proteindelivery. In some embodiments, the polynucleotide is used in genetherapy.

In some embodiments, the protein is a recombinant protein.

In some embodiments, the protein is obtained from bacterial, yeast ormammalian cell culture. In some embodiments, the protein is obtainedfrom E. coli, Pichia pastoris or Chinese Hamster Ovary cells.

In some embodiments, the protein is glycosylated.

In some embodiments, the protein is a fusion protein. In someembodiments, the protein is an Fc fusion protein.

In some embodiments, the polynucleotide is in the form of a vector.

In some embodiments, the polynucleotide is in the form of a viralvector.

In some embodiments, the vector is an adeno-associated viral (AAV)vector, retroviral vector, lentiviral vector or adenoviral vector. Inpreferred embodiments, the vector is an adeno-associated viral (AAV)vector.

In some embodiments, the vector is an AAV2 vector.

In some embodiments, the vector is an AAV9 vector.

In some embodiments, the vector is an AAV8 vector.

In another aspect, the invention provides a vector for use in treatingor reducing the risk of heart disease, wherein the vector comprises apolynucleotide as disclosed herein. In another aspect, the inventionprovides a vector for use in reducing the risk of heart failure, whereinthe vector comprises a polynucleotide as disclosed herein. In anotheraspect, the invention provides a vector for use in preserving cardiacmuscle cell viability, wherein the vector comprises a polynucleotide asdisclosed herein.

In some embodiments, the vector is a viral vector.

In some embodiments, the vector is an adeno-associated viral (AAV)vector, retroviral vector, lentiviral vector or adenoviral vector. Inpreferred embodiments, the vector is an adeno-associated viral (AAV)vector.

In some embodiments, the vector is an AAV2 vector.

In some embodiments, the vector is an AAV9 vector.

In some embodiments, the vector is an AAV8 vector.

In some embodiments, the protein is administered parenterally. In someembodiments, the protein is administered intramyocardially. In someembodiments, the polynucleotide is administered parenterally. In someembodiments, the polynucleotide is administered intramyocardially.

In another aspect, the invention provides a pharmaceutical compositioncomprising the protein as disclosed herein and a pharmaceuticallyacceptable vehicle and/or excipient.

In another aspect, the invention provides a pharmaceutical compositioncomprising the vector as disclosed herein and a pharmaceuticallyacceptable vehicle and/or excipient.

In some embodiments, the composition is formulated for injection. Inpreferred embodiments, the composition is formulated for intracardiac orintravenous administration.

In another aspect, the invention provides the pharmaceutical compositiondisclosed herein for use in treating or reducing the risk of heartdisease. In another aspect, the invention provides the pharmaceuticalcomposition disclosed herein for use in reducing the risk of heartfailure. In another aspect, the invention provides the pharmaceuticalcomposition disclosed herein for use in preserving cardiac muscle cellviability.

In another aspect, the invention provides a protein selected from thegroup consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or apolynucleotide encoding therefor, for use as medicament.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FunSel, an in vivo selection procedure to identify novel cardiactherapeutics for myocardial infarction (MI)

A. Schematic representation of the pGi vector plasmid used for theSecretome library generation. B. Outline of the functional selection(FunSel) procedure to identify cardioactive factors after myocardialinfarction (MI). Briefly, Pools of 50 vectors are used to transduce theheart left ventricle; each vector enters a different cardiomyocyte. MIis then induced, which kills most of the cardiomyocytes, however notthose that express a cardioprotective factor. After 3 weeks, barcode DNAis recovered from the heart by PCR amplification and frequency ofvectors is determined by next generation sequencing (NGS). The number ofsequencing reads of vectors with or without infarction are thencompared; enriched factors are those that exert cardioprotectiveactivity. C. Cumulative results obtained from the in vivo competitivescreening of the 1198 AAV vectors of the library, organized into 24AAV9-pools, each one composed of 50 inserts of similar size. Thefrequency of each factor recovered from the heart after infarction (n=9mice per pool in the MI group) is reported as a ratio to the frequencyof the same factor in the absence of the selective treatment (n=6 miceper pool in the Control group), as detected by NGS barcodequantification 3 weeks after AAV pools injection. D. Cumulative resultsobtained from the in vivo screening of the top 200 factors enriched inthe first round of selection. From this additional round of selection 3factors, namely Chrdl1, Fam3c and Fam3b, never associated with cardiacfunction before, were chosen for further individual investigation. Foldenrichment is expressed as Z-score over undamaged hearts (0=noselection) (Z>1.96 or Z<−1.96; p<0.05).

FIG. 2. Overexpression of Chrdl1, Fam3c and Fam3b in a mouse model ofmyocardial infarction

A-B. Echocardiographic analysis to evaluate heart function at 15, 30 and60 days post-MI in adult CD1 mice transduced with AAV2/9-Chrdl1,AAV2/9-Fam3c, AAV2/9-Fam3b or an AAV2/9-control (1×10¹¹ vg/animal; n=8animals per group). Cardiac overexpression of the three secreted factorsmediated by AAV9 vectors preserves left ventricle ejection fraction(LVEF) (A) and reduces cardiac dilation (Vd—left ventricle volume indiastole) (B), if compared with AAV2/9-control animals. C. Two monthspost-MI, mice were sacrificed for histological analysis and scar sizewas quantified after Masson's trichrome staining (infarct size expressedas % of left ventricle). Heart treatment with the three factors markedlyreduced scar size. D. Measurement of CM cross-sectional area after wheatgerm agglutinin (WGA) staining of heart sections in AAV2/9-Chrdl1,AAV2/9-Fam3c, AAV2/9-Fam3b or AAV2/9-control treated mice, 60 days afterMI. Treatment with the three factors counteracted post-MI myocardialcell hypertrophy, indicative of improved cardiac function. Data areshown as mean±SEM; * P<0.05; ** P<0.01; *** P<0.001.

FIG. 3. Chrdl1, Fam3c and Fam3b counteract the pattern of geneexpression associated with pathological LV remodelling

A-D. Real-time PCR quantification of cardiac expression levels of α-MHC(A), β-MHC (B), SERCA2a (C) and RYR2 (D) genes in non-infarcted animalsand in AAV2/9-control or AAV2/9-Chrdl1, AAV2/9-Fam3c or AAV2/9-Fam3btreated hearts 60 days after MI. Values are normalized for GAPDH andexpressed as fold over untreated (n=6). Each of the three factors waseffective in preserving the heart against pathological LV remodelling.Untr: untreated controls, Ctr: controls with MI. Data are shown asmean±SEM; * P<0.05; ** P<0.01.

FIG. 4. AAV9-mediated cardiac overexpression of Chrdl1, Fam3c and Fam3breduces cell death and promotes beneficial autophagy after MI

A. Quantification of apoptosis by the evaluation of TUNEL-positivenuclei (% of total) in the infarct border zone. Animals were transducedwith AAV2/9 vectors expressing Chrdl1, Fam3c or Fam3b after MI. TUNELstaining of apoptotic cells was performed 2 days later on frozen heartsection (n=5 per group). All three factors were effective at protectingmyocardial cells against apoptotic death. An AAV2/9 vector notexpressing any proteins was used as a control for these experiments.B-C. Induction of autophagy was evaluated by analyzing LC3 proteinlipidation (conversion from LC3-1 to LC3-11) in the left ventricles oftransduced hearts harvested 2 days after MI. Representative western blot(B) and densitometric analysis. Both AAV2/9-Chrdl1 and AAV2/9-Fam3c werecapable to induce autophagy (C). D. To analyze the autophagic flux invivo, adult CD1 mice were transduced with AAV2/9-mRFP-EGFP-LC3 togetherwith AAV2/9s expressing Chrdl1, Fam3c, Fam3b or AAV2/9-Control (n=5 pergroup). Autophagosomes (yellow) and autolysosomes (red) were quantified2 days after MI on frozen heart sections by counting yellow and red LC3+dots. Both AAV2/9-Chrdl1 and AAV2/9-Fam3c markedly increased autophagicflux in vivo. Data are mean±SEM; *P<0.05; **P<0.01.

FIG. 5. Therapeutic effect of circulating Chrdl1, Fam3c and Fam3b aftermyocardial infarction

A. Outline of the strategy to assess therapeutic efficacy of the factorsreleased into the circulation from the liver, to mimic systemicadministration of recombinant proteins. AAV2/8 vectors expressingChrdl1, Fam3c and Fam3b under the control of the hepatocyte-specifichuman α-1 antitrypsin (hAAT) promoter were injected into the liver viadirect intraparenchimal inoculation (n=7; 3 injection sites per liverfor a total of 5×10¹¹ vg/animal); 7 days later the animals underwent MIby coronary artery ligation. B. At 7 days from AAV2/8 intraparenchimaldelivery, circulating Chrdl1, Fam3c and Fam3b proteins were detected inanimal sera by western blotting. C-D. At 15, 30 and 60 after MI,circulating Chrld1, Fam3c and Fam3b preserved left ventricular ejectionfraction (EF) (C), and reduced cardiac dilation (measured as diastolicvolume, μl) (D). E. Scar size was measured as % of LV by Masson'strichrome stain. All three factors reduced infarct size. Data are shownas mean±SEM; *P<0.05; **P<0.01; ***P<0.001.

FIG. 6. Recombinant Chrdl1, Fam3c and Fam3b protect cardiomyocytesagainst doxorubicin-induced cell death

Rat cardiomyocytes were treated with the indicated doses of doxorubicinand then administered with 100 ng/ml of the indicated factors. After 20hours, apoptosis was measured by assessing the levels of caspase 3/7activation. All three recombinant proteins protected the cells. Data areshown as mean±SEM; *P<0.05; **P<0.01.

FIG. 7. Effect of Chrdl1 in preventing fibroblast activation and cardiacfibrosis after myocardial damage

A. Experimental outline to evaluate the effects of Chrdl1 onTgfβ1-induced conversion of primary adult fibroblasts into pro-fibroticmyofibroblasts. B. Adult fibroblasts were treated with increasingdosages of Tgfβ1 (1-10-50 ng/ml) in the presence or absence ofrecombinant Chrdl1 (100 ng/ml). Three days after treatments, Col1a1 andα-Sma levels were evaluated by immunofluorescence. C. The modulation ofChrdl1 on the fibrotic response induced by MI was confirmed in vivo intransgenic Collα1(I)-EGFP mice. AAV2/9-Chrdl1 overexpressing heartsshowed a reduction in the fibrotic scar and in the levels of Col1a1 andα-Sma activation. D. Quantification of the transcriptional levels ofCol1a1, α-Sma, Tgfβ1 and MMP9 by qPCR in heart tissue of CD1 mice 3 daysafter MI. Collectively, these results indicate that Chrdl1 exerts ananti-fibrotic effect after MI. Data are shown as mean±SEM; *P<0.05;**P<0.01.

FIG. 8. Expression of Chrdl1, Fam3c and Fam3b protects mice fromdoxorubicin-induced cardiac toxicity and death.

CD1 mice (n=8) were injected intramyocardially with AAV9 vectorsexpressing the three proteins (Chrdl1, Fam3c or Fam3b), followed bytreatment with doxorubicin for 80 days using infusion pumps. A.Kaplan-Meier survival curve showing strong protective activity of theChrdl1, Fam3c and Fam3b against drug-induced death. B. and C. LeftVentricle (LV) ejection fraction and end-systolic internal diameter,LVEF and LVIDs respectively, at different times during treatment. Thedata show remarkable cardioprotective effect of treatment with any ofthe three factors. Data are shown as mean±SEM; *P<0.05; **P<0.01.

DETAILED DESCRIPTION OF THE INVENTION

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including” or “includes”; or “containing” or“contains”, and are inclusive or open-ended and do not excludeadditional, non-recited members, elements or steps. The terms“comprising”, “comprises” and “comprised of” also include the term“consisting of”.

Proteins

Chrdl1 is a Bone Morphogenetic Protein (BMP) extracellular inhibitor,mainly expressed in mesenchyme-derived cell types, pericytes in theretina and in neural cells (Sakuta, H., et al. Ventroptin: a BMP-4antagonist expressed in a double-gradient pattern in the retina. Science293, 111-115 (2001); Nakayama, N., et al. A novel chordin-like proteininhibitor for bone morphogenetic proteins expressed preferentially inmesenchymal cell lineages. Dev Biol 232, 372-387 (2001); Chandra, A., etal. Neurogenesin-1 differentially inhibits the osteoblasticdifferentiation by bone morphogenetic proteins in C2C12 cells. BiochemBiophys Res Commun 344, 786-791 (2006); Coffinier, C., Tran, U.,Larrain, J. & De Robertis, E. M. Neuralin-1 is a novel Chordin-relatedmolecule expressed in the mouse neural plate. Mech Dev 100, 119-122(2001)).

The Chrdl1 name derives from its sequence similarity with Chordin,another BMP inhibitor identified as a factor dorsalizing Xenopus embryo.Chrdl1 has a spatiotemporal expression pattern distinct from Chordin,but both genes contain cysteine-rich units designed procollagen repeats(CRs), which are also present in a variety of extracellular matrixproteins. CR1 and CR3 are responsible for Chrdl1-BMPs binding. Theprotein binds with high affinity to BMP4 and with less affinity to BMP5,BMP6 and BMP7.

Fam3b and Fam3c are two members of the family with sequence similarity3, FAM3 (Zhu, Y., et al. Cloning, expression, and initialcharacterization of a novel cytokine-like gene family. Genomics 80,144-150 (2002)). Fam3b, also known as PANDER, is highly expressed in thepancreas, where it participates in regulation of glucose homeostasis andβ-cell function (Robert-Cooperman, C. E., Wilson, C. G. & Burkhardt, B.R. PANDER KO mice on high-fat diet are glucose intolerant yet resistantto fasting hyperglycemia and hyperinsulinemia. FEBS Lett 585, 1345-1349(2011); Robert-Cooperman, C. E., et al. Targeted disruption ofpancreatic-derived factor (PANDER, FAM3B) impairs pancreatic beta-cellfunction. Diabetes 59, 2209-2218 (2010); Yang, J., et al. Mechanisms ofglucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B)in pancreatic beta-cells. Diabetes 54, 3217-3228 (2005)).

Fam3c, also known as ILEI, is ubiquitously expressed. It inducesinner-ear cell proliferation (Pilipenko, V. V., Reece, A., Choo, D. I. &Greinwald, J. H., Jr. Genomic organization and expression analysis ofthe murine Fam3c gene. Gene 335, 159-168 (2004)), modulates osteogenicdifferentiation (Bendre, A., Buki, K. G. & Maatta, J. A. Fam3c modulatesosteogenic differentiation by down-regulating Runx2. Differentiation 93,50-57 (2017)) and has a role in epithelial-mesenchymal transition (EMT)during cancer progression (Waerner, T., et al. ILEI: a cytokineessential for EMT, tumor formation, and late events in metastasis inepithelial cells. Cancer Cell 10, 227-239 (2006); Lahsnig, C., et al.ILEI requires oncogenic Ras for EMT of hepatocytes and liver carcinomaprogression. Oncogene 28, 638-650 (2009)). Restoration of its levels inthe liver of obese diabetic mice improves insulin resistance and reducesfatty liver (Chen, Z., et al. Hepatic Activation of the FAM3C-HSF1-CaMPathway Attenuates Hyperglycemia of Obese Diabetic Mice. Diabetes 66,1185-1197 (2017); Chen, Z., et al. FAM3C activates HSF1 to suppresshepatic gluconeogenesis and attenuate hyperglycemia of type 1 diabeticmice. Oncotarget 8, 106038-106049 (2017)).

An example amino acid sequence of Chrld1 is SEQ ID NO: 1 (human)—UniProtID: Q9BU40-1.

(SEQ ID NO: 1) MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGYFCGEETMPVYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLVTRTTLSQWKIFTEGEAQISQMCSSRVCRTELEDLVKVLYLER SEKGHC

An example amino acid sequence of Fam3c is SEQ ID NO: 2 (human)—UniProtID: Q92520-1.

(SEQ ID NO: 2) MRVAGAAKLVVAVAVFLLTFYVISQVFEIKMDASLGNLFARSALDTAARSTKPPRYKCGISKACPEKHFAFKMASGAANVVGPKICLEDNVLMSGVKNNVGRGINVALANGKTGEVLDTKYFDMWGGDVAPFIEFLKAIQDGTIVLMGTYDDGATKLNDEARRLIADLGSTSITNLGFRDNWVFCGGKGIKTKSPFEQHIKNNKDTNKYEGWPEVVEMEGCIPQKQD

An example amino acid sequence of Fam3b is SEQ ID NO: 3 (human)—UniProtID: P58499-1.

(SEQ ID NO: 3) MRPLAGGLLKVVFVVFASLCAWYSGYLLAELIPDAPLSSAAYSIRSIGERPVLKAPVPKRQKCDHWTPCPSDTYAYRLLSGGGRSKYAKICFEDNLLMGEQLGNVARGINIAIVNYVTGNVTATRCEDMYEGDNSGPMTKFIQSAAPKSLLFMVTYDDGSTRLNNDAKNAIEALGSKEIRNMKERSSWVFIAAKGLELPSEIQREKINHSDAKNNRYSGWPAEIQIEGCIPKERS

An example nucleotide sequence encoding Chrld1 (human) is SEQ ID NO: 4(Chrld1)—Seq ID: NM_001143981.1 (coding sequence).

(SEQ ID NO: 4) ATGAGAAAAAAGTGGAAAATGGGAGGCATGAAATACATCTTTTCGTTGTTGTTCTTTCTTTTGCTAGAAGGAGGCAAAACAGAGCAAGTAAAACATTCAGAGACATATTGCATGTTTCAAGACAAGAAGTACAGAGTGGGTGAGAGATGGCATCCTTACCTGGAACCTTATGGGTTGGTTTACTGCGTGAACTGCATCTGCTCAGAGAATGGGAATGTGCTTTGCAGCCGAGTCAGATGTCCAAATGTTCATTGCCTTTCTCCTGTGCATATTCCTCATCTGTGCTGCCCTCGCTGCCCAGAAGACTCCTTACCCCCAGTGAACAATAAGGTGACCAGCAAGTCTTGCGAGTACAATGGGACAACTTACCAACATGGAGAGCTGTTCGTAGCTGAAGGGCTCTTTCAGAATCGGCAACCCAATCAATGCACCCAGTGCAGCTGTTCGGAGGGAAACGTGTATTGTGGTCTCAAGACTTGCCCCAAATTAACCTGTGCCTTCCCAGTCTCTGTTCCAGATTCCTGCTGCCGGGTATGCAGAGGAGATGGAGAACTGTCATGGGAACATTCTGATGGTGATATCTTCCGGCAACCTGCCAACAGAGAAGCAAGACATTCTTACCACCGCTCTCACTATGATCCTCCACCAAGCCGACAGGCTGGAGGTCTGTCCCGCTTTCCTGGGGCCAGAAGTCACCGGGGAGCTCTTATGGATTCCCAGCAAGCATCAGGAACCATTGTGCAAATTGTCATCAATAACAAACACAAGCATGGACAAGTGTGTGTTTCCAATGGAAAGACCTATTCTCATGGCGAGTCCTGGCACCCAAACCTCCGGGCATTTGGCATTGTGGAGTGTGTGCTATGTACTTGTAATGTCACCAAGCAAGAGTGTAAGAAAATCCACTGCCCCAATCGATACCCCTGCAAGTATCCTCAAAAAATAGACGGAAAATGCTGCAAGGTGTGTCCAGGTAAAAAAGCAAAAGAAGAACTTCCAGGCCAAAGCTTTGACAATAAAGGCTACTTCTGCGGGGAAGAAACGATGCCTGTGTATGAGTCTGTATTCATGGAGGATGGGGAGACAACCAGAAAAATAGCACTGGAGACTGAGAGACCACCTCAGGTAGAGGTCCACGTTTGGACTATTCGAAAGGGCATTCTCCAGCACTTCCATATTGAGAAGATCTCCAAGAGGATGTTTGAGGAGCTTCCTCACTTCAAGCTGGTGACCAGAACAACCCTGAGCCAGTGGAAGATCTTCACCGAAGGAGAAGCTCAGATCAGCCAGATGTGTTCAAGTCGTGTATGCAGAACAGAGCTTGAAGATTTAGTCAAGGTTTTGTACCTGGAGAGATCTGAAAAGGGCCACTGTTAG

An example nucleotide sequence encoding Fam3c is SEQ ID NO: 5(Fam3c)—Seq ID: NM_014888.3 (coding sequence).

(SEQ ID NO: 5) ATGAGGGTAGCAGGTGCTGCAAAGTTGGTGGTAGCTGTGGCAGTGTTTTTACTGACATTTTATGTTATTTCTCAAGTATTTGAAATAAAAATGGATGCAAGTTTAGGAAATCTATTTGCAAGATCAGCATTGGACACAGCTGCACGTTCTACAAAGCCTCCCAGATATAAGTGTGGGATCTCAAAAGCTTGCCCTGAGAAGCATTTTGCTTTTAAAATGGCAAGTGGAGCAGCCAACGTGGTGGGACCCAAAATCTGCCTGGAAGATAATGTTTTAATGAGTGGTGTTAAGAATAATGTTGGAAGAGGGATCAATGTTGCCTTGGCAAATGGAAAAACAGGAGAAGTATTAGACACTAAATATTTTGACATGTGGGGAGGAGATGTGGCACCATTTATTGAGTTTCTGAAGGCCATACAAGATGGAACAATAGTTTTAATGGGAACATACGATGATGGAGCAACCAAACTCAATGATGAGGCACGGCGGCTCATTGCTGATTTGGGGAGCACATCTATTACTAATCTTGGTTTTAGAGACAACTGGGTCTTCTGTGGTGGGAAGGGCATTAAGACAAAAAGCCCTTTTGAACAGCACATAAAGAACAATAAGGATACAAACAAATATGAAGGATGGCCTGAAGTTGTAGAAATGGAAGGATGCATCCCCCAGAAGCAAGACTAA

An example nucleotide sequence encoding Fam3b is SEQ ID NO: 6(Fam3b)—Seq ID: NM_058186.3 (coding sequence).

(SEQ ID NO: 6) ATGCGCCCATTGGCTGGTGGCCTGCTCAAGGTGGTGTTCGTGGTCTTCGCCTCCTTGTGTGCCTGGTATTCGGGGTACCTGCTCGCAGAGCTCATTCCAGATGCACCCCTGTCCAGTGCTGCCTATAGCATCCGCAGCATCGGGGAGAGGCCTGTCCTCAAAGCTCCAGTCCCCAAAAGGCAAAAATGTGACCACTGGACTCCCTGCCCATCTGACACCTATGCCTACAGGTTACTCAGCGGAGGTGGCAGAAGCAAGTACGCCAAAATCTGCTTTGAGGATAACCTACTTATGGGAGAACAGCTGGGAAATGTTGCCAGAGGAATAAACATTGCCATTGTCAACTATGTAACTGGGAATGTGACAGCAACACGATGTTTTGATATGTATGAAGGTGATAACTCTGGACCGATGACAAAGTTTATTCAGAGTGCTGCTCCAAAATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACTGAATAACGATGCCAAGAATGCCATAGAAGCACTTGGAAGTAAAGAAATCAGGAACATGAAATTCAGGTCTAGCTGGGTATTTATTGCAGCAAAAGGCTTGGAACTCCCTTCCGAAATTCAGAGAGAAAAGATCAACCACTCTGATGCTAAGAACAACAGATATTCTGGCTGGCCTGCAGAGATCCAGATAGAAGGCTGCATACCCAAAGAAC GAAGCTGA

Any other polynucleotide coding for the above proteins is comprised inthe present invention.

Activity of the proteins of the disclosure and fragments thereof can bereadily determined by the skilled person. For example, suitable in vitroassays include: (a) protection from hydrogen peroxide- ordoxorubicin-induced cell death, for example using TUNEL assays orCaspase activation assays; (b) induction of autophagy, for example theassaying the formation of LC3-positive autophagy vesicles; and/or (c)for Chrdl1: reduction of BMP and TGFbeta activities, for exampleactivation of aSMA expression in cardiac fibroblasts upon treatment withrecombinant TGFbeta, or reduction of SMAD 1/5/8 phosphorylation upontreatment with recombinant BMP4.

The present inventors have surprisingly discovered a previously unknownrole of Chrdl1, Fam3c and Fam3b in promoting cardiomyocyte survival,which indicates their therapeutic activity to counteract ischemia andother forms of cardiac damage and thus prevent heart failure.

According to the present invention, Chrdl1, Fam3c and Fam3b effectivelyincrease cardiac function and reduce infarct size after intracardiacinjection of viral vectors expressing these factors.

While the present inventors do not wish to be bound to theory ormechanism of action, it is believed that the proteins Chrdl1, Fam3c andFam3b exert therapeutic effects in the heart by preventing cardiomyocyteapoptosis and inducing cardiomyocytes autophagy. Together, this resultsin prevention of cardiac function, reduction of fibrosis and leftventricle pathological remodelling and induction of a beneficialexpression pattern of genes associated with pathological cardiacremodeling, such as increase in Serca2a (Sarcoplasmic/endoplasmicreticulum calcium ATPase 2a) and RYR2 (Ryanodin receptor 2) andpreserved α-myosin-heavy-chain (αMHC) to β-myosin-heavy-chain (βMHC)ratio.

A mechanism through which Chrdl1, Fam3c and Fam3b exert cardioprotectiveeffects on ischemic heart is the preservation of myocytes viability bypreventing apoptotic cell death, for example after intracardiacinjection of viral vectors expressing the factors.

Chrdl1 and Fam3c may promote beneficial autophagy to counteractcardiomyocytes cell death after myocardial infarction.

Chrdl1, Fam3c and Fam3b may preserve myocyte viability by preventingapoptotic cell death after doxorubicin treatment.

An additional positive effect of Chrdl1 is to prevent cardiac fibroblastactivation and cardiac fibrosis.

The heart is an organ that is incapable of significant regenerationduring the adult life, thus integrity of cardiac myocytes is maintainedby autophagy, a mechanism that permits renewal of specific intracellularcomponents, including mitochondria. This mechanism is of particularrelevance after myocardial infarction, since sudden ischemia, orischemia followed by reperfusion as after percutaneousrevascularization, causes significant damage to mitochondria, whichstart using oxygen to generate damaging chemical species (Yelton, D M.,Hausenloy D J. Myocardial reperfusion injury. N. Engl. J. Med. 357,1121-1135 (2007); Gustafsson A B., Gottlieb R A. Circ. Res. 104(2),150-158 (2009)). Therefore, autophagy and apoptosis are highlyinterconnected, with the former mechanism becoming activated afterinsults to remove damaged cellular organelles as a protective responseto avoid apoptotic cell death.

The Chrdl1, Fam3c and Fam3b may therefore be used for cardioprotection,thus reducing the risk of heart disease or heart failure. Thecardioprotection may be by preserving cardiac muscle cell viability.

Protein Delivery

As an alternative to the delivery of polynucleotides, the proteins ofthe invention may be delivered by direct protein delivery.

Proteins may be administered directly to a subject. Is some embodiments,the protein is a fusion protein, preferably a fusion with a secondprotein capable of increasing the lifespan of the protein in thesubject. For example, the protein may be an immunoglobulin Fcdomain-fusion protein.

Protein delivery may be via vector delivery (Cai, Y. et al. (2014) Elife3: e01911; Maetzig, T. et al. (2012) Curr. Gene Ther. 12: 389-409).Vector delivery involves the engineering of viral particles (e.g.lentiviral particles) to comprise the proteins to be delivered to acell. Accordingly, when the engineered viral particles enter a cell aspart of their natural life cycle, the proteins comprised in theparticles are carried into the cell.

Protein delivery (Gaj, T. et al. (2012) Nat. Methods 9: 805-7) may alsobe achieved, for example, by utilising a vehicle (e.g. liposomes).

Polynucleotide

Polynucleotides of the invention may comprise DNA or RNA, preferablyDNA. They may be single-stranded or double-stranded. It will beunderstood by a skilled person that numerous different polynucleotidescan encode the same polypeptide as a result of the degeneracy of thegenetic code. In addition, it is to be understood that skilled personsmay, using routine techniques, make nucleotide substitutions that do notaffect the polypeptide sequence encoded by the polynucleotides of theinvention to reflect the codon usage of any particular host organism inwhich the polypeptides of the invention are to be expressed.

The nucleotide sequences of the invention disclosed herein may compriseor lack stop codons at their 3′ end, for example depending on theirposition in a bicistronic vector. Thus, the present disclosureencompasses the SEQ ID NOs disclosed herein with the stop codons presentor absent.

The polynucleotides may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or lifespan of the polynucleotides of the invention.

Polynucleotides such as DNA polynucleotides may be producedrecombinantly, synthetically or by any means available to those of skillin the art. They may also be cloned by standard techniques.

Longer polynucleotides will generally be produced using recombinantmeans, for example using polymerase chain reaction (PCR) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking the target sequence which it is desired toclone, bringing the primers into contact with mRNA or cDNA obtained froman animal or human cell, performing a polymerase chain reaction underconditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixturewith an agarose gel) and recovering the amplified DNA. The primers maybe designed to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable vector.

Vectors

A vector is a tool that allows or facilitates the transfer of an entityfrom one environment to another.

In one aspect, the invention provides a vector comprising apolynucleotide of the invention. In preferred embodiments, the vector isa viral vector. In some embodiments, the vector is an adeno-associatedviral (AAV) vector, retroviral vector, lentiviral vector or adenoviralvector, preferably an AAV vector.

Adeno-Associated Viral (AAV) Vectors

In one aspect, the invention provides an AAV vector comprising apolynucleotide of the invention.

Preferably, the AAV vector is in the form of an AAV vector particle.

In some embodiments, the AAV vector particle comprises an AAV2 genome.In some embodiments, the AAV vector particle comprises an AAV9 genome.In some embodiments, the AAV vector particle comprises an AAV8 genome.

In some embodiments, the AAV vector particle comprises AAV9 capsidproteins. In some embodiments, the AAV vector particle comprises AAV8capsid proteins.

In some embodiments, the AAV vector particle comprises an AAV2 genomeand AAV9 capsid proteins (AAV2/9). In other embodiments, the AAV vectorparticle comprises an AAV2 genome and AAV8 capsid proteins (AAV2/8).

Methods of preparing and modifying viral vectors and viral vectorparticles, such as those derived from AAV, are well known in the art.

The AAV vector may comprise an AAV genome or a fragment or derivativethereof.

AAV is known to be capable of packaging genomes up to 5.2 kb in size(Dong, J.-Y. et al. (1996) Human Gene Therapy 7: 2101-2112).

An AAV genome is a polynucleotide sequence, which may encode functionsneeded for production of an AAV particle. These functions include thoseoperating in the replication and packaging cycle of AAV in a host cell,including encapsidation of the AAV genome into an AAV particle.Naturally occurring AAVs are replication-deficient and rely on theprovision of helper functions in trans for completion of a replicationand packaging cycle. Accordingly, the AAV genome of the AAV vector ofthe invention is typically replication-deficient.

The AAV genome may be in single-stranded form, either positive ornegative-sense, or alternatively in double-stranded form. The use of adouble-stranded form allows bypass of the DNA replication step in thetarget cell and so can accelerate transgene expression.

The AAV genome may be from any naturally derived serotype, isolate orclade of AAV. Thus, the AAV genome may be the full genome of a naturallyoccurring AAV. As is known to the skilled person, AAVs occurring innature may be classified according to various biological systems.

Commonly, AAVs are referred to in terms of their serotype. A serotypecorresponds to a variant subspecies of AAV which, owing to its profileof expression of capsid surface antigens, has a distinctive reactivitywhich can be used to distinguish it from other variant subspecies.Typically, a virus having a particular AAV serotype does not efficientlycross-react with neutralising antibodies specific for any other AAVserotype.

AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10 and AAV11, and also recombinant serotypes, such as Rec2 andRec3, recently identified from primate brain. Any of these AAV serotypesmay be used in the invention.

In some embodiments, the AAV vector particle is an AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2 or Rec3 AAVvector particle.

In some embodiments, the AAV is an AAV1, AAV2, AAV5, AAV7, AAV8 or AAV9serotype.

In some embodiments, the AAV is an AAV9 or AAV8 serotype.

In some embodiments, the AAV is an AAV9 serotype. In other embodiments,the AAV is an AAV8 serotype.

The capsid protein may be a mutant capsid protein such as disclosed inWO 2008/124724, which is herein incorporated by reference.

In some embodiments, the AAV vector comprises an AAV8 capsid with anY733F mutation.

Reviews of AAV serotypes may be found in Choi et al. (2005) Curr. GeneTher. 5: 299-310 and Wu et al. (2006) Molecular Therapy 14: 316-27. Thesequences of AAV genomes or of elements of AAV genomes including ITRsequences, rep or cap genes for use in the invention may be derived fromthe following accession numbers for AAV whole genome sequences:Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3BNC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCCVR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263,AY629583; Bovine AAV NC_005889, AY388617.

AAV may also be referred to in terms of clades or clones. This refers tothe phylogenetic relationship of naturally derived AAVs, and typicallyto a phylogenetic group of AAVs which can be traced back to a commonancestor, and includes all descendants thereof. Additionally, AAVs maybe referred to in terms of a specific isolate, i.e. a genetic isolate ofa specific AAV found in nature. The term genetic isolate describes apopulation of AAVs which has undergone limited genetic mixing with othernaturally occurring AAVs, thereby defining a recognisably distinctpopulation at a genetic level.

The skilled person can select an appropriate serotype, clade, clone orisolate of AAV for use in the invention on the basis of their commongeneral knowledge. For instance, the AAV5 capsid has been shown totransduce primate cone photoreceptors efficiently as evidenced by thesuccessful correction of an inherited colour vision defect (Mancuso etal. (2009) Nature 461: 784-7).

The AAV serotype determines the tissue specificity of infection (ortropism) of an AAV. Accordingly, preferred AAV serotypes for use in AAVsadministered to patients in accordance with the invention are thosewhich have natural tropism for or a high efficiency of infection oftarget cells within the heart.

Typically, the AAV genome of a naturally derived serotype, isolate orclade of AAV comprises at least one inverted terminal repeat sequence(ITR). An ITR sequence acts in cis to provide a functional origin ofreplication and allows for integration and excision of the vector fromthe genome of a cell. In preferred embodiments, one or more ITRsequences flank the nucleotide sequences encoding the protein of theinvention. The AAV genome typically also comprises packaging genes, suchas rep and/or cap genes which encode packaging functions for an AAVparticle. The rep gene encodes one or more of the proteins Rep78, Rep68,Rep52 and Rep40 or variants thereof. The cap gene encodes one or morecapsid proteins such as VP1, VP2 and VP3 or variants thereof. Theseproteins make up the capsid of an AAV particle. Capsid variants arediscussed below.

A promoter will be operably linked to each of the packaging genes.Specific examples of such promoters include the p5, p19 and p40promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76:5567-5571). For example, the p5 and p19 promoters are generally used toexpress the rep gene, while the p40 promoter is generally used toexpress the cap gene.

As discussed above, the AAV genome used in the AAV vector of theinvention may therefore be the full genome of a naturally occurring AAV.For example, a vector comprising a full AAV genome may be used toprepare an AAV vector or vector particle in vitro. However, while such avector may in principle be administered to patients, this will rarely bedone in practice. Preferably, the AAV genome will be derivatised for thepurpose of administration to patients. Such derivatisation is standardin the art and the invention encompasses the use of any known derivativeof an AAV genome, and derivatives which could be generated by applyingtechniques known in the art. Derivatisation of the AAV genome and of theAAV capsid are reviewed in: Coura and Nardi (2007) Virology Journal 4:99, and in Choi et al. and Wu et al., referenced above.

Derivatives of an AAV genome include any truncated or modified forms ofan AAV genome which allow for expression of a transgene from an AAVvector of the invention in vivo. Typically, it is possible to truncatethe AAV genome significantly to include minimal viral sequence yetretain the above function. This is preferred for safety reasons toreduce the risk of recombination of the vector with wild-type virus, andalso to avoid triggering a cellular immune response by the presence ofviral gene proteins in the target cell.

Typically, a derivative will include at least one inverted terminalrepeat sequence (ITR), preferably more than one ITR, such as two ITRs ormore. One or more of the ITRs may be derived from AAV genomes havingdifferent serotypes, or may be a chimeric or mutant ITR. A preferredmutant ITR is one having a deletion of a trs (terminal resolution site).This deletion allows for continued replication of the genome to generatea single-stranded genome which contains both coding and complementarysequences, i.e. a self-complementary AAV genome. This allows for bypassof DNA replication in the target cell, and so enables acceleratedtransgene expression.

The one or more ITRs will preferably flank the nucleotide sequenceencoding the protein of the invention at either end. The inclusion ofone or more ITRs is preferred to aid concatamer formation of the vectorof the invention in the nucleus of a host cell, for example followingthe conversion of single-stranded vector DNA into double-stranded DNA bythe action of host cell DNA polymerases. The formation of such episomalconcatamers protects the vector construct during the life of the hostcell, thereby allowing for prolonged expression of the transgene invivo.

In preferred embodiments, ITR elements will be the only sequencesretained from the native AAV genome in the derivative. Thus, aderivative will preferably not include the rep and/or cap genes of thenative genome and any other sequences of the native genome. This ispreferred for the reasons described above, and also to reduce thepossibility of integration of the vector into the host cell genome.Additionally, reducing the size of the AAV genome allows for increasedflexibility in incorporating other sequence elements (such as regulatoryelements) within the vector in addition to the transgene.

The following portions could therefore be removed in a derivative of theinvention: one inverted terminal repeat (ITR) sequence, the replication(rep) and capsid (cap) genes. However, in some embodiments, derivativesmay additionally include one or more rep and/or cap genes or other viralsequences of an AAV genome. Naturally occurring AAV integrates with ahigh frequency at a specific site on human chromosome 19, and shows anegligible frequency of random integration, such that retention of anintegrative capacity in the vector may be tolerated in a therapeuticsetting.

Where a derivative comprises capsid proteins i.e. VP1, VP2 and/or VP3,the derivative may be a chimeric, shuffled or capsid-modified derivativeof one or more naturally occurring AAVs. In particular, the inventionencompasses the provision of capsid protein sequences from differentserotypes, clades, clones, or isolates of AAV within the same vector(i.e. a pseudotyped vector).

Chimeric, shuffled or capsid-modified derivatives will be typicallyselected to provide one or more desired functionalities for the AAVvector. Thus, these derivatives may display increased efficiency of genedelivery, decreased immunogenicity (humoral or cellular), an alteredtropism range and/or improved targeting of a particular cell typecompared to an AAV vector comprising a naturally occurring AAV genome,such as that of AAV2. Increased efficiency of gene delivery may beeffected by improved receptor or co-receptor binding at the cellsurface, improved internalisation, improved trafficking within the celland into the nucleus, improved uncoating of the viral particle andimproved conversion of a single-stranded genome to double-stranded form.Increased efficiency may also relate to an altered tropism range ortargeting of a specific cell population, such that the vector dose isnot diluted by administration to tissues where it is not needed.

Chimeric capsid proteins include those generated by recombinationbetween two or more capsid coding sequences of naturally occurring AAVserotypes. This may be performed for example by a marker rescue approachin which non-infectious capsid sequences of one serotype areco-transfected with capsid sequences of a different serotype, anddirected selection is used to select for capsid sequences having desiredproperties. The capsid sequences of the different serotypes can bealtered by homologous recombination within the cell to produce novelchimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering ofcapsid protein sequences to transfer specific capsid protein domains,surface loops or specific amino acid residues between two or more capsidproteins, for example between two or more capsid proteins of differentserotypes.

Shuffled or chimeric capsid proteins may also be generated by DNAshuffling or by error-prone PCR. Hybrid AAV capsid genes can be createdby randomly fragmenting the sequences of related AAV genes e.g. thoseencoding capsid proteins of multiple different serotypes and thensubsequently reassembling the fragments in a self-priming polymerasereaction, which may also cause crossovers in regions of sequencehomology. A library of hybrid AAV genes created in this way by shufflingthe capsid genes of several serotypes can be screened to identify viralclones having a desired functionality. Similarly, error prone PCR may beused to randomly mutate AAV capsid genes to create a diverse library ofvariants which may then be selected for a desired property.

The sequences of the capsid genes may also be genetically modified tointroduce specific deletions, substitutions or insertions with respectto the native wild-type sequence. In particular, capsid genes may bemodified by the insertion of a sequence of an unrelated protein orpeptide within an open reading frame of a capsid coding sequence, or atthe N- and/or C-terminus of a capsid coding sequence.

The unrelated protein or peptide may advantageously be one which acts asa ligand for a particular cell type, thereby conferring improved bindingto a target cell or improving the specificity of targeting of the vectorto a particular cell population. The unrelated protein may also be onewhich assists purification of the viral particle as part of theproduction process, i.e. an epitope or affinity tag. The site ofinsertion will typically be selected so as not to interfere with otherfunctions of the viral particle e.g. internalisation, trafficking of theviral particle. The skilled person can identify suitable sites forinsertion based on their common general knowledge. Particular sites aredisclosed in Choi et al., referenced above.

The invention additionally encompasses the provision of sequences of anAAV genome in a different order and configuration to that of a nativeAAV genome. The invention also encompasses the replacement of one ormore AAV sequences or genes with sequences from another virus or withchimeric genes composed of sequences from more than one virus. Suchchimeric genes may be composed of sequences from two or more relatedviral proteins of different viral species.

The AAV vector of the invention may take the form of a nucleotidesequence comprising an AAV genome or derivative thereof and a sequenceencoding the protein of the invention.

The AAV particles of the invention include transcapsidated forms whereinan AAV genome or derivative having an ITR of one serotype is packaged inthe capsid of a different serotype. The AAV particles of the inventionalso include mosaic forms wherein a mixture of unmodified capsidproteins from two or more different serotypes makes up the viral capsid.The AAV particle also includes chemically modified forms bearing ligandsadsorbed to the capsid surface. For example, such ligands may includeantibodies for targeting a particular cell surface receptor.

The AAV vector may comprise multiple copies (e.g., 2, 3 etc.) of thenucleotide sequence referred to herein.

In some embodiments, the polynucleotide further comprises one or moreAAV ITRs. In preferred embodiments, the polynucleotide further comprisestwo AAV ITRs. In some embodiments, the polynucleotide comprises an AAVITR at its 5′ end and an AAV ITR at its 3′ end. In some embodiments, theAAV ITRs are AAV2, AAV9 or AAV8 ITRs.

Promoters and Regulatory Sequences

The polynucleotide or vector of the invention may also include elementsallowing for the expression of the nucleotide sequence encoding theprotein of the invention in vitro or in vivo. These may be referred toas expression control sequences. Thus, the polynucleotide or vectortypically comprises expression control sequences (e.g. comprising apromoter sequence) operably linked to the nucleotide sequence encodingthe protein of the invention.

Any suitable promoter may be used, the selection of which may be readilymade by the skilled person. The promoter sequence may be constitutivelyactive (i.e. operational in any host cell background), or alternativelymay be active only in a specific host cell environment, thus allowingfor targeted expression of the transgene in a particular cell type (e.g.a tissue-specific promoter). The promoter may show inducible expressionin response to presence of another factor, for example a factor presentin a host cell. In any event, where the vector is administered fortherapy, it is preferred that the promoter should be functional in thetarget cell background.

In preferred embodiments, the promoter is a liver-specific promoter. Inpreferred embodiments, the promoter is a liver-specific hAAT promoter.

The liver-specific hAAT promoter may confer selective specificity forhepatocytes. When vectors are administered through the portal vein, theinventors have show that each circulating factor, secreted from theliver, can protect the heart after damage.

Suitable promoters include the chicken beta-actin (CBA) promoter,optionally in combination with a cytomegalovirus (CMV) enhancer element.An example promoter for use in the invention is a CAG promoter.

In some embodiments, the promoter is a CMV promoter.

The polynucleotide or vector of the invention may also comprise one ormore additional regulatory sequences which may act pre- orpost-transcriptionally. The regulatory sequence may be part of thenative transgene locus or may be a heterologous regulatory sequence. Thepolynucleotide or vector of the invention may comprise portions of the5′-UTR or 3′-UTR from the native transgene transcript.

Regulatory sequences are any sequences which facilitate expression ofthe transgene, i.e. act to increase expression of a transcript, improvenuclear export of mRNA or enhance its stability. Such regulatorysequences include for example enhancer elements, post-transcriptionalregulatory elements and polyadenylation sites.

Suitable enhancers include the WPRE regulatory element. Suitable poly-Asignals include the Bovine Growth Hormone poly-A signal.

Additional regulatory sequences may be readily selected by the skilledperson.

Method of Administration

A variety of administration routes and techniques may be utilised, amongthem parenteral techniques such as intravenous, intracardiac andintra-arterial injections, catheterisations and the like. Averagequantities of the active agent may vary and in particular should bebased upon the recommendations and prescription of a qualifiedphysician.

The protein, polynucleotide or vector of the invention may beadministered systemically (for example by peripheral vein infusion) ormay be administered locally or regionally.

Preferably, the protein is administered by parenteral route, inparticular intravenous, intraarterial or intramyocardial route.

The administration of the polynucleotide encoding the proteins disclosedherein may be achieved by gene therapy, see for example WO 2013/093870.

According to the present invention, Chrdl1, Fam3c and Fam3b are alsoactive when they reach the infarcted heart through the systemiccirculation.

Pharmaceutical Compositions and Injected Solutions

The medicaments, for example proteins, polynucleotides or vectors, ofthe invention may be formulated into pharmaceutical compositions. Thesecompositions may comprise, in addition to the medicament, apharmaceutically acceptable carrier, diluent, excipient, buffer,stabiliser or other materials well known in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay be determined by the skilled person according to the route ofadministration.

According to the administration route chosen, the compositions may be insolid or liquid form, suitable for oral, parenteral, intravenous orintra-arterial administration. The pharmaceutical composition istypically in liquid form. Liquid pharmaceutical compositions generallyinclude a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,magnesium chloride, dextrose or other saccharide solution, or glycolssuch as ethylene glycol, propylene glycol or polyethylene glycol may beincluded. In some cases, a surfactant, such as pluronic acid (PF68)0.001% may be used.

For injection at the site of affliction, the active ingredient may be inthe form of an aqueous solution which is pyrogen-free, and has suitablepH, isotonicity and stability. The skilled person is well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection or Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included as required.

For delayed release, the medicament may be included in a pharmaceuticalcomposition which is formulated for slow release, such as inmicrocapsules formed from biocompatible polymers or in liposomal carriersystems according to methods known in the art.

Such compositions are well-known in the art, see for example Remington'sPharmaceutical Sciences; last edition, Mack Pub.

Method of Treatment

It is to be appreciated that all references herein to treatment includecurative, palliative and prophylactic treatment; although in the contextof the invention references to preventing are more commonly associatedwith prophylactic treatment. Treatment may also include arrestingprogression in the severity of a disease.

The treatment of mammals, particularly humans, is preferred. However,both human and veterinary treatments are within the scope of theinvention.

The administration regime, dosage and posology will be determined by thephysician according to his experience, the disease to be treated and thepatient's conditions.

The proteins and/or the polynucleotides of the present invention can beadministered either singularly or in combination thereof.

The term “combination”, or terms “in combination”, “used in combinationwith” or “combined preparation” as used herein may refer to the combinedadministration of two or more agents simultaneously, sequentially orseparately.

The term “simultaneous” as used herein means that the agents areadministered concurrently, i.e. at the same time.

The term “sequential” as used herein means that the agents areadministered one after the other.

The term “separate” as used herein means that the agents areadministered independently of each other but within a time interval thatallows the agents to show a combined, preferably synergistic, effect.Thus, administration “separately” may permit one agent to beadministered, for example, within 1 minute, 5 minutes or 10 minutesafter the other.

Variants, Derivatives, Analogues, Homologues and Fragments

In addition to the specific proteins and nucleotides mentioned herein,the invention also encompasses the use of variants, derivatives,analogues, homologues and fragments thereof.

In the context of the invention, a variant of any given sequence is asequence in which the specific sequence of residues (whether amino acidor nucleic acid residues) has been modified in such a manner that thepolypeptide or polynucleotide in question substantially retains itsfunction. A variant sequence can be obtained by addition, deletion,substitution, modification, replacement and/or variation of at least oneresidue present in the naturally-occurring protein.

The term “derivative” as used herein, in relation to proteins orpolypeptides of the invention includes any substitution of, variationof, modification of, replacement of, deletion of and/or addition of one(or more) amino acid residues from or to the sequence providing that theresultant protein or polypeptide substantially retains at least one ofits endogenous functions.

The term “analogue” as used herein, in relation to polypeptides orpolynucleotides includes any mimetic, that is, a chemical compound thatpossesses at least one of the endogenous functions of the polypeptidesor polynucleotides which it mimics.

Typically, amino acid substitutions may be made, for example from 1, 2or 3 to 10 or 20 substitutions provided that the modified sequencesubstantially retains the required activity or ability. Amino acidsubstitutions may include the use of non-naturally occurring analogues.

Proteins used in the invention may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent protein. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity and/or theamphipathic nature of the residues as long as the endogenous function isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include asparagine, glutamine, serine,threonine and tyrosine.

Conservative substitutions may be made, for example according to thetable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R H AROMATIC F W Y

The term “homologue” as used herein means an entity having a certainhomology with the wild type amino acid sequence and the wild typenucleotide sequence. The term “homology” can be equated with “identity”.

A homologous sequence may include an amino acid sequence which may be atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical,preferably at least 95% or 97% or 99% identical to the subject sequence.Typically, the homologues will comprise the same active sites etc. asthe subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the inventionit is preferred to express homology in terms of sequence identity.

A homologous sequence may include a nucleotide sequence which may be atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical,preferably at least 95% or 97% or 99% identical to the subject sequence.Although homology can also be considered in terms of similarity, in thecontext of the invention it is preferred to express homology in terms ofsequence identity.

Preferably, reference to a sequence which has a percent identity to anyone of the SEQ ID NOs detailed herein refers to a sequence which has thestated percent identity over the entire length of the SEQ ID NO referredto.

Homology comparisons can be conducted by eye or, more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate percentagehomology or identity between two or more sequences.

Percentage homology may be calculated over contiguous sequences, i.e.one sequence is aligned with the other sequence and each amino acid inone sequence is directly compared with the corresponding amino acid inthe other sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion in the nucleotide sequence maycause the following codons to be put out of alignment, thus potentiallyresulting in a large reduction in percent homology when a globalalignment is performed. Consequently, most sequence comparison methodsare designed to produce optimal alignments that take into considerationpossible insertions and deletions without penalising unduly the overallhomology score. This is achieved by inserting “gaps” in the sequencealignment to try to maximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps as possible,reflecting higher relatedness between the two compared sequences, willachieve a higher score than one with many gaps. “Affine gap costs” aretypically used that charge a relatively high cost for the existence of agap and a smaller penalty for each subsequent residue in the gap. Thisis the most commonly used gap scoring system. High gap penalties will ofcourse produce optimised alignments with fewer gaps. Most alignmentprograms allow the gap penalties to be modified. However, it ispreferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum percentage homology therefore firstly requiresthe production of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples ofother software that can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al. (1999) ibid—Ch.18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al. (1999) ibid, pages7-58 to 7-60). However, for some applications, it is preferred to usethe GCG Bestfit program. Another tool, called BLAST 2 Sequences is alsoavailable for comparing protein and nucleotide sequences (see FEMSMicrobiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

Although the final percent homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see the user manual for further details). For someapplications, it is preferred to use the public default values for theGCG package, or in the case of other software, the default matrix, suchas BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate percent homology, preferably percent sequence identity. Thesoftware typically does this as part of the sequence comparison andgenerates a numerical result.

“Fragments” of a full length Chrdl1, Fam3c or Fam3b are also variantsand the term typically refers to a selected region of the polypeptide orpolynucleotide that is of interest either functionally or, for example,in an assay. “Fragment” thus refers to an amino acid or nucleic acidsequence that is a portion of a full-length polypeptide orpolynucleotide.

Such variants may be prepared using standard recombinant DNA techniquessuch as site-directed mutagenesis. Where insertions are to be made,synthetic DNA encoding the insertion together with 5′ and 3′ flankingregions corresponding to the naturally-occurring sequence either side ofthe insertion site may be made. The flanking regions will containconvenient restriction sites corresponding to sites in thenaturally-occurring sequence so that the sequence may be cut with theappropriate enzyme(s) and the synthetic DNA ligated into the cut. TheDNA is then expressed in accordance with the invention to make theencoded protein. These methods are only illustrative of the numerousstandard techniques known in the art for manipulation of DNA sequencesand other known techniques may also be used.

The skilled person will understand that they can combine all features ofthe invention disclosed herein without departing from the scope of theinvention as disclosed.

Preferred features and embodiments of the invention will now bedescribed by way of non-limiting examples.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, biochemistry, molecularbiology, microbiology and immunology, which are within the capabilitiesof a person of ordinary skill in the art. Such techniques are explainedin the literature. See, for example, Sambrook, J., Fritsch, E. F. andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 andperiodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNAIsolation and Sequencing: Essential Techniques, John Wiley & Sons;Polak, J. M. and McGee, J. O'D. (1990) In Situ Hybridization: Principlesand Practice, Oxford University Press; Gait, M. J. (1984)Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley,D. M. and Dahlberg, J. E. (1992) Methods in Enzymology: DNA StructuresPart A: Synthesis and Physical Analysis of DNA, Academic Press. Each ofthese general texts is herein incorporated by reference.

EXAMPLES Example 1 FunSel, an In Vivo Selection Procedure to IdentifyNovel Cardiac Therapeutics for Myocardial Infarction

We recently developed FunSel (1,2), a novel procedure for the in vivofunctional identification of novel therapeutic factors againstdegenerative conditions that here we applied with the aim to identifyfactors ensuring cardiac protection after myocardial infarction (MI).This is based on the use of adeno-associated virus (AAV) vectors, whichare exquisite tools for highly efficient cardiac gene transfer (3).

In brief, we generated an arrayed library of genes that corresponds tothe secretome, defined as the subset of proteins secreted into theextracellular environment of a cell. By a computational approach (4),2033 unique proteins have been identified in the genome, bearing asignal peptide and lacking any transmembrane domain or intracellularlocalization signal and thus potentially secreted from the cells. Thesize of the AAV genome, limiting cloning to 4.5 kb, and the availabilityof cDNA clones, limited the number of cDNAs suitable for cloning to1198. The coding region of these genes was individually cloned into apAAV pGi backbone plasmid under the control of the constitutive CMV IEpromoter and confirmed by sequencing. A unique 10-nt barcode, which canbe PCR-amplified and sequenced, univocally identifies each clone (FIG.1A).

FunSel is based on the following strategy: a pool of AAV plasmids fromthe library, each one coding for a specific factor and being identifiedby a unique barcode, was used for the batch production of AAV serotype 9(AAV9) vectors (for a total of 24 pools composed by 50 factors ofsimilar size each). Eight-week-old CD1 mice (n=9 animals for each AAV9pool), were subjected to myocardial infarction induced by permanentligation of the left descending coronary artery, which in this settingrepresents the selective stimulus. Immediately after MI, each AAV9 poolof vectors was injected in vivo into the left ventricle (LV)peri-infarctual region at a multiplicity by which each vector, inprinciple, enters a different cell (10{circumflex over ( )}¹⁰ viralgenomes per animal). After three weeks, vector inserts were recoveredfrom the surviving LV tissue and the frequency of each vector wasdetermined by next generation sequencing (NGS) of the barcodes andcompared to that found in control animals (n=6 animals per pool)injected with the same AAV9 pool, but not submitted to MI. Afterinfarction most myocyte cells die, however when a cell expresses aprotective factor then selectively survives, therefore enrichment for abarcode indicates positive gene selection (beneficial effect), whilereduced representation indicates negative selection (neutral ordetrimental effect) (Experimental scheme in FIG. 1B).

FIG. 1C reports the cumulative results obtained from the in vivoscreening of the 1198 factors. In the graph, the frequency of eachfactor recovered from the heart after MI is reported as a ratio to thefrequency of the same factor in the absence of the selective treatment.Based on Z-score calculation, we selected the top 200 performers and run4 additional screens on these factors (4 pools of 50 vectors). Elevenfactors resulted competitively enriched 1.96 Z-score or more (P<0.05)(FIG. 1D). Supporting the robustness of the FunSel approach, among thehits of this second round of screening we found a few knowncardioprotective factors and unknown factors somehow related tocardiomyocyte biology. These included Mdk, a pleiotropic molecule, whichplays a protective role against cardiac injury and Rln1, awell-described anti-fibrotic agent, able to reduce ROS production,apoptosis and inflammation in the infarcted heart. More notably,however, the top performers included three new proteins for which noinformation is currently available nor study has been performed relativeto cardiac protection. These are Chordin-like 1 (Chrdl1) and two membersof the family with sequence similarity 3, namely Fam3b and Fam3c. Thesefactors were chosen for further individual investigation to assessefficacy and mechanism of action.

Example 2

Efficacy of Chrdl1, Fam3c and Fam3b in Preserving Cardiac Integrity andFunction after Myocardial Infarction in Mice

Based on the FunSel results, we decided to validate and characterize theeffect of the selected top secreted factors (Chrdl1, Fam3c and Fam3b)expressed as individual AAV2/9 vectors upon myocardial infarction inmouse hearts, with the specific purpose to assess the capacity of eachfactor to counteract or reduce ischemic damage and promote cardiacfunction.

Eight-week-old CD1 mice were subjected to MI and, at the same time,injected in the LV peri-infarcted area with AAV2/9 vectors expressingChrdl1, Fam3c, Fam3b or a control empty vector (1×10¹¹ vg/animal; n=8per group). Our previous experience indicates that this procedureresults in efficient myocardial transduction and month-long expressionof the transgene (1,5). Cardiac function of the animals was monitored byechocardiography at 15, 30 and 60 days post MI.

As reported in FIG. 2, AAV2/9-mediated overexpression of Chrdl1, Fam3cor Fam3b successfully preserved LV ejection fraction (LVEF) (FIG. 2A) ofinfarcted mice when compared to control treated animals. The LVEF valuesstarted to be remarkably improved at 15 days post MI and were maintainedovertime (60 days after MI: AAV9-Chrdl1 39.96±2.67%, AAV2/9-Fam3c39.02±2.53% and AAV2/9-Fam3b 32.50±2.70%, compared with 19.86±0.98% forthe animals that received the control vector, P<0.001 for alltreatments). At both 30 and 60 days post MI, the diastolic LV volume(Vd) was significantly larger, as expected when heart failure starts tooccur, in control animals if compared with treated mice (FIG. 2B) (60days after MI: AAV2/9-Chrdl1 101.22±10.31p1, AAV2/9-Fam3c 107.31±8.85p1and AAV2/9-Fam3b 115.16±11.03p1, compared with 168.64±7.47p1 for theanimals that received the control vector, P<0.001 for all treatments).

When myocardial infarction occurs, cardiac fibroblasts proliferate,differentiate into myofibroblasts and produce extracellular matrix tocreate a scar to replace the gap generated by cardiomyocyte loss. Thisoften results in myocardial stiffness and leads to pathologicalremodelling of the left ventricle, dilatation and dysfunction.Morphometric analysis on trichromic-stained heart sections at day 60indicated that AAV2/9-Chrdl1, AAV2/9-Fam3c and AAV2/9-Fam3b-treated miceshowed significant preservation of LV contractile tissue and reductionof the fibrotic area (infarct size: AAV2/9-Chrdl1 6.88±1.60%,AAV2/9-Fam3c 10.19±2.05%, AAV2/9-Fam3b 13.35±1.79%, versus 26.26±2.35%of the LV in control animals; FIG. 2C).

Finally, a wheat germ agglutinin (WGA) staining revealed that nocardiomyocyte hypertrophic response was induced by Chrdl1, Fam3c orFam3b overexpression, since fibre cross sectional area was not increasedin treated animals compared to controls (FIG. 2D).

Taken together, these results indicate that the AAV2/9-mediated cardiacover-expression of Chrdl1, Fam3c or Fam3b after acute ischemia promotesmyocyte cell viability, reduces infarct size and preserves cardiacfunction after myocardial infarction.

Example 3

AAV2/9-Mediated Expression of Chrdl1, Fam3c or Fam3b CounteractsPathological Left Ventricle Remodelling Associated with the Onset ofHeart Failure in Mice

Two months after MI, we investigated also the effect of each secretedfactor on the expression of a set of genes previously associated with LVpathological remodelling (consisting in overexpression of β-myosin heavychain (pMHC) and decreased levels of α-myosin heavy chain (αMHC),Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) andryanodine receptor 2 (RYR2)).

Total RNA was extracted from LV tissue and analysed by qRT-PCR usingTaqMan probes specific for the investigated genes. Consistent withechocardiographic and morphometric observations, AAV2/9-Chrdl1,AAV2/9-Fam3c and AAV2/9-Fam3b counteracted the characteristic pattern ofgene expression typically associated with pathological LV remodellingobserved in AAV2/9-control mice, reducing the level of β-MHC andincreasing those of α-MHC, SERCA2a and RYR2 (FIG. 3, panel A-D).

Example 4

All Three Factors Preserve Tissue Viability Protecting Cardiomyocytesfrom Cell Death and Both Chrdl1 and Fam3c Promote Cardiac BeneficialAutophagy after MI

So far, the available information on Chrdl1, Fam3c and Fam3b factors isscanty and certainly cannot explain why these factors exert acardioprotective effect after myocardial infarction. Therefore, we setout to explore possible biological mechanisms that might mediate theiractivity. Since the FunSel approach is based on factor selection basedon cardiomyocyte survival, a first set of experiments was performed bytesting the levels of cell death from apoptosis, which is usuallymassive at two days after coronary artery occlusion (6), in the heartsof infarcted adult CD1 mice treated with the three AAV vectors, comparedto controls (1×10¹¹ vg/animal; n=5 per group). Fam3b, Fam3c and, inparticular, Chrdl1, were extremely effective in preventing apoptoticcell death in the infarcted hearts, as assessed by nuclear TUNEL(TdT-mediated dUTP nick-end labelling) staining on snap frozen heartsection 2 days after MI (% of positive TUNEL nuclei: AAV2/9-Chrdl14.01±1.21%, AAV2/9-Fam3c 10.33±1.43%, AAV2/9-Fam3b 19.83±3.01%, versus30.67±4.38% of in control animals) (quantification in FIG. 4A).

The heart is an organ that is incapable of significant regenerationduring the adult life, thus integrity of cardiac myocytes is maintainedby autophagy, a mechanism that permits renewal of specific intracellularcomponents, in particular mitochondria. This mechanism is of particularrelevance after myocardial infarction, since sudden ischemia, orischemia followed by reperfusion as after percutaneousrevascularization, causes significant damage to mitochondria, whichstart using oxygen to generate damaging chemical species (7). Notsurprising, therefore, autophagy and apoptosis are highlyinterconnected, with the former mechanism becoming activated afterdamage to remove damaged cellular organelles as a protective response toavoid apoptotic cell death (8).

To evaluate induction of autophagy after acute cardiac ischemia weinjected another group of adult, infarcted CD1 mice with AAV2/9 vectorsexpressing Chrdl1, Fam3c, Fam3b or a control vector (1×10¹¹ vg/animal;n=5 per treatment). Two days after MI, we found increased conversion ofthe soluble LC3-1 protein to lipid-bound LC3-11, in particular in thehearts of Chrdl1 and Fam3c treated mice, which associated with theformation of autophagosomes (representative blots and quantification forAAV2/9-Chrdl1, AAV2/9-Fam3c, AAV9-Fam3b and AAV2/9-control hearts inFIGS. 4B and 4C, respectively).

To directly visualize the autophagic flux in the infarcted hearts, wepreviously generated an AAV2/9 vector expressing the monomeric redfluorescent protein (mRFP)-enhanced green fluorescent protein (EGFP)tandem fluorescent-tagged LC3 protein, derived from the ptfLC3 plasmid,in which green, but not red, fluorescence is sensitive to the pHdifference between the neutral autophagosome and the acidic autolysosome(9). This vector was administered, together with AAV2/9-Chrdl1,AAV2/9-Fam3c, AAV2/9-Fam3b or AAV2/9-control (1×10¹¹ vg/animal; n=5 pertreatment), immediately after MI. Two days later, the number of yellow,LC3-positive vesicles, and, in particular, of those showing only redfluorescence, was significantly increased in the LV perinfarct region ofhearts injected with AAV9-Chrdl1 and Fam3c, indicating that these twofactors stimulate the autophagic flux in vivo (quantification of yellowand red puncta for each treatment are reported in FIG. 4D).

Taken together, these results indicate that the AAV2/9-mediated cardiacover-expression of Chrdl1 Fam3c, Fam3b preserves cardiac myocyteviability by preventing apoptotic cell death and, in particular Fam3cand Chrdl1, promoting cardiac beneficial autophagy.

Example 5

Circulating Chrdl1, Fam3c and Fam3b, Produced and Secreted by the LiverUpon AAV8-Mediated Tissue Specific Expression, Counteract PathologicalLeft Ventricle Remodelling after Myocardial Infarction

To assess whether circulating Chrdl1, Fam3b and Fam3c are active oncereaching the heart from the circulation, as opposed to beingendogenously expressed using viral vectors, we designed a strategy bywhich each of the three factors is expressed by the liver and secretedinto the circulation before myocardial infarction (FIG. 5A). In moredetail, we performed an intraparenchimal injection in adult CD1 mice(5×10¹¹ vg/animal; n=6 per group) with AAV vectors serotype 8 (AAV2/8),which selectively transfers genes into liver cells; in these vectors,the factor is expressed under the control of the human α-1 antitrypsin(hAAT) promoter, which ensures specific expression in hepatocytes only(10). Seven days after administration, when the liver was activelyproducing and releasing dosable amounts of each factor in thecirculation (FIG. 5B), myocardial infarction was induced by ligating theleft descendant coronary artery.

As reported in FIG. 5C, AAV2/8-mediated liver production of Chrdl1,Fam3c or Fam3b successfully preserved LV ejection fraction (LVEF) ofinfarcted mice compared to control treated animals. The LVEF valuesstarted to be remarkably improved at 15 days post MI and were maintainedovertime (60 days after MI: AAV2/8-Chrdl1 28.77±1.66%, AAV2/8-Fam3c28.09±1.61% and AAV8-Fam3b 31.22±1.40%, compared to 20.05±1.47% for theanimals that received the control vector). Two months after MI, asexpected, the diastolic LV volume was significantly larger in controlanimals compared to treated mice (FIG. 5D) (60 days after MI:AAV2/8-Chrdl1 150.2±10.2 μl, AAV2/8-Fam3c 137.9±16.8 μl and AAV2/8-Fam3b134.7±9.2 μl, compared to 193.2±11.6 μl for the animals that receivedthe control vector).

Finally, also in this experiment, morphometric analysis ontrichromic-stained heart sections at day 60 indicated that AAV8-Chrdl1,AAV2/8-Fam3c and AAV2/8-Fam3b-treated mice showed significant reductionof the fibrotic area (infarct size: AAV2/8-Chrdl1 13.6±3.1%,AAV2/8-Fam3c 15.0±3.4%, AAV2/8-Fam3b 13.2±2.7%, versus 28.7±3.3% of theLV in control animals; FIG. 5E).

Taken together these results prove that each circulating factor,therapeutically expressed from the liver, protect cardiomyocytes fromischemic damage and improve cardiac function after MI. This representsan efficacy test preliminary to the injection of Chrdl1, Fam3c and Fam3bas recombinant proteins.

Example 6 Recombinant Chrdl1, Fam3c and Fam3b Protect CardiomyocytesAgainst Doxorubin-Induced Cell Death

To evaluate the potential effect of Chrdl1, Fam3c and Fam3b inpreserving cell viability upon toxic damage, the correspondingrecombinant proteins were tested on primary neonatal rat ventricularcardiomyocytes treated with the chemotherapeutic drug doxorubicin (FIG.6).

The treatment with 100 ng/ml of Chrdl1, Fam3c or Fam3b recombinantproteins significantly counteracted caspase 3/7 activation (as a measureof apoptotic cell death), after 20 hours of doxorubicin treatment (1 and1.5 μM).

Example 7

Chrdl1 Prevents Fibroblast Activation and Cardiac Fibrosis afterMyocardial Infarction

When MI occurs, cardiac fibroblasts proliferate, differentiate intomyofibroblasts and stimulate collagen deposition to create a scar toreplace the gap generated by cardiomyocyte loss. Of interest, infarctedhearts overexpressing Chrdl1 not only had very small scars but also didnot underwent pathological remodelling and dilatation at two monthsafter MI. This was suggestive of a specific effect of Chrdl1 on scarformation, in addition to that on cardiomyocyte survival.

Transforming growth factor-β1 (Tgfβ1), which is expressed at high levelsin the scar after MI, is a key inductor of collagen deposition anddifferentiation of fibroblasts into myofibroblasts (11). To evaluatewhether Chrdl1 was able to modulate fibroblast transdifferentiationinduced by Tgfβ1, primary adult mouse cardiac fibroblasts were treatedfor 3 days with different Tgfβ1 dosages (1-10-50 ng/ml) in the presenceor absence of recombinant Chrdl1 (100 ng/ml) (FIG. 7A). Tgfβ1 induced amassive and dose-dependent increase of collagen α-1(I) (Col1α1) andα-Sma expression while Chrdl1 blunted this effect (FIG. 7B).

To additionally investigate the effect of Chrdl1 in the fibroticresponse in vivo, the hearts of Collα1(1)-EGFP mice (a transgenic mousemodel in which EGFP is only expressed in fibroblasts (12)) weretransduced with AAV2/9-control or AAV2/9-Chrdl1 and MI was induced. Thehearts of Col1a1-eGFP mice overexpressing Chrdl1 showed a significantlyattenuated cardiac fibrosis and reduced collagen1α1 and α-SMA expression(FIG. 7C). These data were also confirmed by q-PCR quantifying theCol1α1, α-SMA, Tgfβ1 and MMP9 transcript levels 3 days after infarctionin CD1 mice (FIG. 7D).

Example 8

Expression of Chrdl1, Fam3c and Fam3b Protects Mice fromDoxorubicin-Induced Cardiac Toxicity and Death

Despite their efficacy as anticancer medicines, anthracyclines(including doxorubicin) can induce both acute and chronic cardiotoxicity(Swain, S. M. et al. (2003) Cancer 97: 2869-2879). In particular,cumulative doses of these drugs can cause left ventricular systolicdysfunction and heart failure. The reported incidence of ventriculardysfunction as a consequence of treatment with these drugs can be ashigh as approximately 10% of patients (Cardinale, D. (2015) Circulation131: 1981-1988), with the vast majority of cases occurring within thefirst year of treatment. Currently, there is no standard therapy toprevent anthracycline-induced cardiotoxicity (Zamorano, J. L. et al.(2016) Eur Heart J 37: 2768-2801).

Six-week old, female C57/BL6 mice were injected intramyocardially with30 μl of AAV9 vector preparations expressing Chrdl1, Fam3c or Fam3busing a 30G-needle syringe. One week later, doxorubicin was administeredintraperitoneally at days 0, 2, 5, 8, 10 and 12 at the concentration of4 mg/kg (cumulative dose: 24 mg/kg), according to an establishedprotocol for chronic treatment (Li M. et al. (2018) Circulation 138:696-711). Mice were followed with echocardiography at weeks 0, 6 and 8.

Each of Chrdl1, Fam3c and Fam3b showed strong protective activityagainst drug-induced death (FIG. 8A). A remarkable cardioprotectiveeffect is observed following treatment with any of the three factors, asshown by the significant protection both against deterioration in LeftVentricle (LV) ejection fraction (FIG. 8B), and deleterious effects onLV internal diameters (FIG. 8C).

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All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedisclosed agents, compositions, uses and methods of the invention willbe apparent to the skilled person without departing from the scope andspirit of the invention. Although the invention has been disclosed inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the disclosedmodes for carrying out the invention, which are obvious to the skilledperson are intended to be within the scope of the following claims.

1. A protein selected from the group consisting of Chrdl1, Fam3c, Fam3band fragments thereof, or a polynucleotide encoding therefor, for use intreating or reducing the risk of heart disease.
 2. A protein selectedfrom the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof,or a polynucleotide encoding therefor, for use in preserving cardiacmuscle cell viability.
 3. The protein or polynucleotide for useaccording to claim 1 or 2, wherein the protein comprises an amino acidsequence that has at least 70% identity to SEQ ID NO: 1, 2 or
 3. 4. Theprotein or polynucleotide for use according to claim 1 or 2, wherein thepolynucleotide comprises a nucleotide sequence that has at least 70%identity to SEQ ID NO: 4, 5 or
 6. 5. The protein or polynucleotide foruse according to any one of claim 1, 3 or 4, wherein the heart diseaseis associated with cardiac ischemia or loss of cardiomyocytes.
 6. Theprotein or polynucleotide for use according to any one of claim 1 or3-5, wherein the heart disease is selected from myocardial infarction;the consequences of myocardial infarction; reperfusion-injury afterpercutaneous coronary intervention; myocarditis; hypertension; cardiactoxic damage (in particular, by chemotherapy); or cardiomyopathy.
 7. Theprotein or polynucleotide for use according to any preceding claim,wherein the heart is protected from myocardial infarction; cardiacfunction is preserved after myocardial infarction or percutaneouscoronary intervention; or fibrosis after infarction is reduced.
 8. Theprotein or polynucleotide for use according to any preceding claim,wherein heart failure is prevented.
 9. The protein for use according toany preceding claim, wherein the protein is glycosylated.
 10. Theprotein for use according to any preceding claim, wherein the protein isa fusion protein, preferably an Fc fusion protein.
 11. Thepolynucleotide for use according to any one of claims 1-8, wherein thepolynucleotide is in the form of a vector, optionally a viral vector,preferably an adeno-associated viral (AAV) vector.
 12. A vector for usein treating or reducing the risk of heart disease, wherein the vectorcomprises a polynucleotide as defined in any one of claim 1-8 or
 11. 13.The vector for use according to claim 12, wherein the vector is a viralvector, optionally wherein the viral vector is an adeno-associated viral(AAV) vector.
 14. A pharmaceutical composition comprising the protein asdefined in any one of claims 1-10 and a pharmaceutically acceptablevehicle and/or excipient.
 15. The pharmaceutical composition accordingto claim 14, wherein the composition is formulated for injection.
 16. Apharmaceutical composition comprising the vector as defined in claim 12or 13 and a pharmaceutically acceptable vehicle and/or excipient. 17.The pharmaceutical composition according to any one of claims 14-16 foruse as defined in any one of claims 1-13.